Matrix Assisted Laser Desorption Ionization (MALDI) has become an important ionization technique for use in mass spectrometry. MALDI ion sources are typically configured to produce ions in vacuum pressure that is lower than 10xe2x88x924 torr. Ions are produced in MALDI ionization by impinging a pulse of laser light onto a target on which a sample solution has been deposited with an appropriate matrix. The resulting ions produced from a MALDI laser pulse are directed into a mass spectrometer where they are mass to charge analyzed. Time-Of-Flight (TOF) mass analyzers are particularly well suited to mass to charge analyze MALDI generated ions. Ions produced from a MALDI pulse in the TOF vacuum region are accelerated into the TOF flight tube and mass analyzed. Techniques such as delayed extraction or reverse acceleration have been employed to improve the resolution when acquiring low vacuum pressure MALDI TOF mass spectra. TOF mass analyzers are capable of separating and detecting ions over a wide mass to charge range, which is essential when analyzing higher molecular weight compounds. MALDI ion sources have also been interfaced to other mass spectrometer types including Fourier Transform Mass Spectrometers (FTMS) and three dimensional quadrupole ion traps (Ion Traps).
Several recipes are available for optimizing a sample and MALDI matrix combination for a given laser wavelength. Typically a nitrogen laser may be used with a DHB matrix. The matrix is chosen to absorb the laser wavelength and transfer the laser power to the matrix to achieve rapid heating of the sample. The rapid heating desorbs and ionizes the sample that was initially dissolved and dried in the matrix solution and a portion of the sample molecules are ionized in the desorption process. To prepare a sample for MALDI ionization, sample solution and matrix solution are combined, deposited on a MALDI probe and dried prior to insertion of the probe into the MALDI ion source. Various conductive and dielectric materials such as glass, metal, silicon and plastics have been configured for use as the MALDI probe substrate. Hydrophobic substrate materials have been used to avoid spreading and thinning of the sample and matrix solution when it is deposited on the probe. It is desirable to concentrate the sample in as small a volume as possible on the MALDI probe to increase the sample ion yield per laser pulse. The MALDI probe substrate should not react with the sample, contribute minimum background peaks in the mass spectrum and allow sufficient binding of sample and matrix to prevent sample loss during MALDI probe handling. When conditioned silicon surfaces are used as MALDI targets, the use of a matrix solution can be eliminated. In some of the embodiments of the invention described below, the additional constraint of using a dielectric MALDI probe material allows the configuration of MALDI probe targets positioned within multipole ion guides or ion funnels causing minimum distortion of Electric fields.
Ions produced from MALDI ion sources configured in the low vacuum pressure region of TOF mass analyzers can be pulsed directly into the TOF MS flight tube for mass analysis. This configuration minimizes any constraint on the mass to charge range that can be analyzed but may limit the resolving power and mass measurement accuracy that can be achieved. Ions that are produced from a MALDI matrix have an uncorrelated energy and spatial spread in the pulsing region of a TOF mass analyzer, resulting in reduced resolving power and mass measurement accuracy in TOF ion mass to charge analysis. Although delayed extraction or reverse field extraction of MALDI produced ions has reduced the effects of ion energy and spatial spread, the techniques have a limit as to how much improvement can be achieved. Also delayed extraction must be carefully tuned to minimize distortion of ion signal intensities in the mass to charge range of interest. The kinetic energy spread of MALDI produced ions also reduces the ion transport and capture efficiency in FTMS and ion trap mass analyzers resulting in decreased sensitivity. Mass to charge selection and fragmentation experiments known as MS/MS experiments may be achieved by using MALDI post source decay or by the configuration of gas collision cells in TOF mass analyzer flight tubes. Ion fragmentation and MS/MS TOF experiments have been achieved using these TOF techniques at some sacrifice to resolving power, mass measurement accuracy and, in some configurations, sensitivity. In an effort to improve mass to charge measurement, resolving power, mass to charge selection precision and efficiency and fragmentation efficiency in MS/MS analysis of MALDI produced samples, MALDI ion sources have been configured in atmospheric pressure and in intermediate vacuum pressure regions of mass analyzers.
Introducing MALDI samples into an atmospheric (AP) or intermediate vacuum pressure (IP) MALDI ion source facilitates sample handling by eliminating the need to load MALDI samples into low vacuum pressure. Laiko et al. in U.S. Pat. No. 5,965,884 and in Anal. Chem. 2000, 72, 652-657 describe the configuration of an atmospheric pressure MALDI Ion source interfaced to an orthogonal pulsing TOF mass analyzer. Krutchincsky et al. J. Am. Soc. Mass Spectrom 2000, 11, 493-504, describe the configuration of MALDI ion source in the second vacuum pumping stage of a hybrid quadrupole/quadrupole/orthogonal pulsing TOF (QTOF) mass analyzer that includes an atmospheric pressure Electrospray ion source. In the atmospheric and vacuum pressure MALDI mass spectrometers described, the ions traverse at least one multipole ion guide prior to being pulsed into the TOF mass analyzer. The mass to charge range of ions that can be analyzed is limited to the range of mass to charge values that can be transmitted with stable ion trajectories through the downstream ion guides. Ion guides positioned in the first or second vacuum pumping stages have pressures maintained sufficiently high to cause multiple ion to neutral background collisions. Elevated background pressures in multipole ion guides cause damping of ion kinetic energies as the ions traverse an ion guide length. The energy damping creates a primary ion beam with a narrow energy spread and a controlled average kinetic energy. Ion mass to charge selection and collisional induced dissociation fragmentation can be achieved in single or multiple ion guide assemblies prior to TOF mass to charge analysis. The upstream ion kinetic energy damping processes result in improved TOF resolving power and ion mass to charge measurement accuracy in orthogonal pulsing TOF. MALDI ionization at atmospheric and intermediate vacuum pressure may yield differences in ion populations when compared with low vacuum pressure MALDI ionization. Neutral to ion collisions occurring in atmospheric pressure and intermediate vacuum pressure MALDI ion source regions reduce the internal energy of the newly formed ion, minimizing post source decay. Subsequent MS/MS functions can be conducted in downstream multipole ion guides, ion traps, FTMS censor TOF-TOF mass analyzers is user controlled through selected experimental methods. The decoupling of the MALDI ionization, ion mass to charge selection, ion fragmentation and subsequent ion mass to charge analysis steps allows independent optimization of each analytical step.
Laiko et al. describe the configuration of a sample MALDI probe positioned near the orifice into vacuum of an API TOF MS instrument so that a portion of the ions produced can be transported into vacuum. A DC field is applied between the MALDI sample target and the orifice into vacuum to direct ions toward the orifice. A gas flow directed over the probe surface was added to push ions produced near the probe surface toward the orifice into vacuum. Laiko reports that substantial sensitivity losses occurred when using the atmospheric pressure MALDI ion source compared with a MALDI ion source configured in the pulsing region of a TOF mass analyzer. Most of the loss of signal was attributed to inefficient ion transport into vacuum. The resulting mass spectrum also included peaks of sample ions clustered with matrix molecules. This clustering may occur due to the condensing of neutral matrix molecules with sample ions in the free jet expansion into vacuum. Krutchinsky et al. describes the configuration of a MALDI probe in the second vacuum stage of a four vacuum stage QTOF where the MALDI target is positioned upstream of the entrance lens orifice to an RF only quadrupole ion guide operating in the second vacuum pumping stage of the QTOF mass analyzer. An additional quadrupole ion guide was added in the second vacuum stage to improve the Electrospray (ES) ion transport efficiency when the MALDI target was removed. Good sensitivities were achieved with MALDI and ES ion sources with the configuration reported. The use of a MALDI ion source operated in vacuum pressure requires that the MALDI target be loaded into vacuum. This constrains the size and shape of the MALDI probe and requires that additional components be added to minimize a decrease in performance of the atmospheric pressure ion sources configured together in the same instrument. Cleaning the vacuum pressure MALDI ion source region requires vacuum venting in the intermediate vacuum pressure stages, causing instrument downtime.
One embodiment of the invention, improves the transport efficiency of ions produced in an atmospheric pressure ion source and reduces or eliminates the number of neutral matrix molecules entering vacuum. The elimination of neutral matrix related molecules from entering vacuum prevents condensation of the matrix molecules with the sample ions in the free jet expansion into vacuum. This eliminates cluster matrix related peaks in the acquired mass spectra. The invention improves the ion transport efficiency into vacuum by reducing the initial atmospheric pressure MALDI (AP MALDI) ion energy spread through ion to neutral collisional damping or focusing of the ion trajectories to the centerline of a multipole ion guide or ion funnel operated at atmospheric pressure with RF voltage applied. AP MALDI generated ions are focused along the centerline and directed to the orifice into vacuum in the ion guides or ion funnels operated at atmospheric pressure. Ions can be trapped and some degree of mass to charge selection achieved using mulipole ion guides at atmospheric pressure. Multipole ion guides have been used to efficiently damp the trajectories of ions and transport ions in intermediate vacuum pressures as have been reported in U.S. Pat. No. 5,652,427 (Whitehouse et al ""427), U.S. Pat. No. 6,011,259 (Whitehouse et al. ""259) and U.S. Pat. No. 4,963,736 (Douglas et al.). RF only Ion Funnels operated in intermediate vacuum pressure regions of 1 to 2 torr in API MS instruments have been reported by Belov et al., J. Am. Soc. Mass Spectrom 2000, 11, 19-23 and U.S. Pat. No. 6,107,628. Although Douglas et al. achieves effective collisional energy damping in intermediate vacuum pressures they report a severe decrease in ion signal for background pressures above 70 millitorr. Miniature quadrupole mass spectrometers configured for use as vacuum pressure gauges as described by R. J. Ferran and S. Boumsellek, J. Vac. Sci. Technol., A 14(3), May/June 1996 exhibit a decrease in ion signal intensity for pressures which have a mean free path longer than the miniature quadrupole rod dimensions. The reported upper practical operating pressure is the point where the ion to neutral collisional mean free path is roughly equal to the length of the quadrupole ion guide described. Whitehouse et. al. ""427 report the operation of a multipole ion guide in background pressures of hundreds of millitorr with little or no loss of ion signal intensity over the entire operating background pressure range. The efficiency of ion transmission through multipole ion guides or ion funnels is maximized by moving ions through the ion guide with axial electric fields and/or directed neutral gas flow. In the present invention, ions are transmitted through a multipole ion guide or ion funnel configured in an atmospheric or vacuum pressure region where multiple collisions occur between ions and neutral background gas molecules during transmission. Ion transmission losses are minimized by providing axial DC voltages and/or gas dynamics to move MALDI generated ions through the entrance RF fringing fields and through the ion guide or ion funnel length. In one embodiment of the invention, atmospheric pressure or vacuum pressure MALDI ions are generated directly in the RF ion trapping field of the multipole ion guides or ion funnels thus avoiding ion scattering losses due to entrance fringing fields entirely.
Ion mobility analyzers have been interfaced with mass spectrometers to allow separation of ions due to differences in ion mobility prior to conducting ion mass to charge analysis. Such a hybrid instrument allows the separation of ions having the same mass to charge value but different collisional cross sections to be analytically separated in mass spectrometric measurements. Coupling ion mobility separation with mass to charge analysis of ions provides additional information regarding the tertiary structure of a molecule or ion. U.S. Pat. No. 5,905,258 (Klemmer) and U.S. Pat. No. 5,936,242 (De La Mora) describe ion mobility analyzers interfaced to mass spectrometers. Klemmer describes a mobility analyzer interfaced to an orthogonal pulsing TOF mass analyzer. De La Mora and Klemmer describe ion mobility analyzers that employ DC electric fields and gas flow to separate ions by their mobility. Unlike the prior art which uses DC only electric fields in a background gas to separate ions due to different ion mobility, the invention enables ion mobility separation from AP MALDI generated ions to occur within a multipole ion guide prior to conducting mass to charge analysis. In the invention, ions are exposed to RF as well as DC electric fields as they traverse the ion guide length. Ion collisions with neutral background gas causes translational energy damping of ion trajectories to the centerline and spatial separation of ions with different ion mobility along the ion guide axis. By radially trapping ions with RF fields and directing the ions in the axial direction with DC fields, the sampling efficiency into the orifice to vacuum after ion mobility separation is improved compared with the ion focusing that can be achieved with DC only electric fields applied in atmospheric pressure as described in the prior.
To facilitate interfacing with higher throughput automated sample preparation and separation systems, the MALDI ion sources must be configured to accommodate a wide range of probe geometries and automated MALDI target sample introduction means. On-line integration of a MALDI ion source with capillary electrophoresis separation systems has been achieved as described by Karger et. al. in U.S. Pat. No. 6,175,112 B1. Sample preparation and separation is being conducted in smaller scale using integrated devices. The current invention is configured to facilitate and optimize the interfacing of an AP MALDI ion source with such integrated sample preparation and sample handing devices and automated MALDI sample target introduction. In one embodiment of the invention, MALDI ionization is conducted from sample deposited on a moving belt positioned to move through a multipole ion guide operated in an atmospheric or vacuum pressure region. The invention allows multiplexed MALDI ionization across parallel sample tracks synchronized with ion pulsing into TOF mass analyzers to increase sample throughput. Improvements in on-line MALDI TOF MS and MS/MSn performance can be achieved according to the invention by conducting MALDI ionization at atmospheric or vacuum pressures from moving belts traversing laterally through a multipole ion guide from which ions can be subsequently mass to charge selected or fragmented prior to a last mass to charge analysis step.
In one embodiment of the invention a multipole ion guide with RF and DC electric fields applied to the poles is operated at atmospheric pressure. A MALDI ion source is configured to operate at atmospheric pressure and deliver ions into the multipole ion guide configured to operate at atmospheric pressure. The transfer of AP MALDI ions into and through the multipole ion guide is aided by directed gas flow and DC electric fields. Ion collisions with the background gas damp the stable ion trajectories toward centerline as the ions traverse the length of the multipole ion guide toward an orifice into vacuum. Axial DC electric fields can also be configured to move the ions through the length of the multipole ion guide toward the orifice into vacuum. Ions focused along the centerline are directed with gas flow and DC electric fields into an orifice into vacuum where the ions are mass to charge analyzed or undergo mass to charge selection and fragmentation steps prior to a final mass to charge analysis step (MS/MSn). Gas flow at the ion guide entrance end is directed along the ion guide axis toward the orifice into vacuum to aid in ion transfer into and through the ion guide along the multipole ion guide centerline. In one embodiment of the invention, a second gas flow is introduced at the ion guide exit end directed axially toward the multipole ion guide entrance end, countercurrent to the first gas flow. Ions move in the axial direction against the second gas flow due to the axial DC electric fields. The second gas flow prevents neutral matrix related molecules from entering vacuum with the MALDI produced ions. Reduction or elimination of neutral contamination molecules avoids recondensation of such molecules with sample ions in the free jet expansion into vacuum.
The orifice into vacuum can be configured as a sharp edged orifice, a nozzle, a dielectric capillary or a conductive capillary. The countercurrent gas and/or the capillary tubes may be heated. The face of the orifice into vacuum comprises a conductive material and can be configured as the exit lens of the multipole ion guide operated at atmospheric pressure. The potential of the orifice into vacuum can be increased higher than the multipole ion guide DC offset or bias potential to trap ions in the ion guide. Ions from several MALDI pulses can be accumulated in the multipole ion guide before release into vacuum in this manner. RF, +/xe2x88x92DC and resonant frequency potentials can be applied to the multipole ion guide to reduce the mass to charge range of stable ion trajectories through the ion guide. Using this method, unwanted contamination or matrix related ions can be eliminated before entering vacuum. In non-trapping mode, the multipole ion guide can be operated as a mobility analyzer where ions generated in an Atmospheric Pressure MALDI pulse separate spatially along the ion guide axis due to different ion mobilities as they traverse the multipole ion guide length. In an alternative embodiment of the invention, one or more additional electrostatic lens can be configured between the multipole ion guide exit and the orifice into vacuum. One of these electrostatic lenses can be split to allow steering of selected ions away from the orifice into vacuum. By timing the switching of voltage levels applied to the steering lens elements while conducting ion mobility separation, selected ions can be allowed to enter the orifice into vacuum. Using this technique, different conformations of the same molecule can be isolated and mass to charge analyzed with MS or MS/MSn experiments to study compound structure.
In an alternative embodiment of the invention, the MALDI probe is configured to place the sample target inside the volume described by the poles of the multipole ion guide operated in atmospheric or vacuum pressure. The MALDI probe and target material may be conductive or dielectric, however, dielectric materials cause minimum distortion of the multipole ion guide RF and DC fields during operation. MALDI ions generated inside the multipole ion guide are trapped in the RF field avoiding the need to transfer ions through RF and DC fringing fields at the ion guide entrance. High capture and transport efficiency can be achieved using this in-multipole ion guide MALDI ion production technique. The MALDI probe can be configured with an array of target samples or be configured as a moving belt to conduct on-line experiments. A moving belt MALDI target can be interfaced on-line or off-line to the outlet of one or more Capillary Electrophoresis (CE) or Liquid Chromatography (LC) columns. The moving belt with the deposited sample and MALDI matrix solution is configured to traverse laterally through the multipole ion guide volume and the sample is ionized near the multipole ion guide centerline as it passes through. The laser beam can be rastered from one sample line to another on the moving belt synchronized with the TOF mass analyzer pulsing to allow multiplexed parallel analysis of several samples with one mass analyzer. This multiple sample analysis technique improves off-line or on-line sample throughput.
In an alternative embodiment of the invention, the MALDI target is configured in an intermediate vacuum pressure region and MALDI produced ions are swept into a multipole ion guide by gas dynamics and applied DC fields. The local gas pressure at the multipole ion guide entrance is maintained higher than the vacuum chamber background gas to aid in sweeping ions into the ion guide entrance minimizing transmission losses due to the ion guide fringing fields. Ions continue to traverse the ion guide length moved by gas dynamics and/or DC fields. Ion to neutral collisions occur as the ions traverse the ion guide length damping the internal and kinetic energies. In one embodiment of the invention the multipole ion guide is configured to extend continuously from one vacuum pumping stage into a subsequent vacuum stage to maximize ion transmission efficiency. The multipole ion guide may be segmented to allow the conducting of ion mass to charge selection and fragmentation analytical functions in the same ion guide volume. This embodiment of the invention improves the ion transfer efficiency of MALDI ions produced in a vacuum pressure region into a mass analyzer. Similar to the atmospheric pressure MALDI ion source embodiment, ion mobility analysis can be conducted on MALDI generated ions in the multipole ion guide configured in an intermediate vacuum pressure region.
MALDI ionization generates positive and negative ions simultaneously. In one embodiment of the invention, a MALDI probe, is configured with the MALDI sample target positioned inside the multipole ion guide. The multipole ion guide may be operated in RF only mode with a DC gradient applied along its axis. The DC gradient is achieved by any number of techniques including but not limited to, configuring the multipole ion guide with segmented, conical or non parallel rods or adding DC electrostatic lens elements external to the multipole rod set which establishes an external axially asymmetric DC field which penetrates to the multipole ion guide centerline. Two mass analyzers are configured to simultaneously accept opposite polarity MALDI generated ions leaving opposite ends of the multipole ion guide. In one embodiment of the invention, the first mass analyzer is operated in positive ion mode and the second analyzer is operated in negative ions mode. Positive MALDI generated ions move along the multipole ion guide axis and exit through one end of the ion guide. The simultaneously produced negative MALDI generated ions move in the opposite direction along the multipole ion guide axis and exit through the opposite end of the ion guide. The positive ions are transferred from the ion guide operated in atmospheric or vacuum pressure and mass to charge analyzed in the first mass to charge analyzer. The negative ions are directed to and mass to charge analyzed in the second mass to charge analyzer.
In an alternative embodiment of the invention, an ion funnel operated with RF and an axial DC fields is configured in place of the multipole ion guide in a MALDI ion source operated in atmospheric or vacuum pressure. The MALDI probe can be configured with the MALDI target positioned inside or outside the ion funnel volume. MALDI produced ions are directed to move axially along the ion funnel using DC fields and directed gas flow. Ion motion in the ion funnel guide is damped due to collisions with background gas resulting in higher ion transport efficiency through the ion funnel exit orifice.
MALDI ion sources operated in atmospheric or vacuum pressure interfaced to multipole ion guides or ion funnels can be configured with but not limited to TOF, TOF-TOF, Ion Trap, Quadrupole, FTMS, hybrid Quadrupole-TOF, magnetic sector, hybrid magnetic sector TOF mass analyzers and other hybrid mass analyzers types.
Other objects, advantages and features of this invention will become more apparent hereinafter.